Recombinant human cxcl13, by R&D Systems - Product details

1

Transwell Assay for B Cell Migration

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Purified human CD19+ B cells were seeded in the upper chamber in 3-μm Trans-well plates (Corning Costar, Cambridge, MA); HUVECs pre-transfected with Empty vector or NK4 were seeded in the bottom chamber.
The Transwell was maintained at 37°C for 24hrs with 5% CO2. The number of B cells that had migrated into the bottom chamber was determined by flow cytometry for CD19+ cells. For CXCL13 neutralization, increasing concentration of neutralizing CXCL13 antibody or an isotype control antibody (10 μg/ml) (R&D Systems) was used. Recombinant human CXCL13 (R&D Systems, Minneapolis, MN) at 500 ng/ml were applied as positive control in the bottom chamber.

Qu P., Wuest T., Min Y., Alevizos I., Young H.A, & Lin P.C. (2020). Natural Killer Cell Transcript 4 promotes the development of Sjögren’s Syndrome via activation of Rap1 on B cells. Journal of autoimmunity, 116, 102559.

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Culturing Human Renal Mesangial Cells

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Human renal mesangial cells (HRMCs) were purchased from JENNIO Biological Technology and cultured in RPMI-1640 medium (HyClone) with 10% fetal bovine serum (FBS, Gibco) at 37°C in 5% CO₂. Cells were treated with recombinant human CXCL13 (R&D Systems, Catalog Number 801-CX-025).

Da Z., Li L., Zhu J., Gu Z., You B., Shan Y, & Shi S. (2016). CXCL13 Promotes Proliferation of Mesangial Cells by Combination with CXCR5 in SLE. Journal of Immunology Research, 2016, 2063985.

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3

Effect of CXCL13 on HRMC Proliferation

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Cell proliferation was monitored using the Cell Counting Kit-8 (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. Aliquots of 100 μL cell suspension were plated into 96-well plates at 1 × 10³ cells per well and cultured in the growth medium for 24 h. Cells were then treated with 0.5 ng/mL recombinant human CXCL13 (R&D Systems, USA). HRMCs were transfected with either 50 nmol/mL miR-155 mimic or 100 nmol/mL siRNA in RPMI 1640 (without serum or antibiotics). The number of viable cells was assessed at 0, 6, 12, 24, and 36 h after treatment. Each well was added with 10 μL CCK-8 solution, incubated for 2.5 h in the dark, and measured the absorbance at 450 nm using a microplate reader (BioTek, USA).

Kong J., Li L., Lu Z., Song J., Yan J., Yang J., Gu Z, & Da Z. (2018). MicroRNA-155 Suppresses Mesangial Cell Proliferation and TGF-β1 Production via Inhibiting CXCR5-ERK Signaling Pathway in Lupus Nephritis. Inflammation, 42(1), 255-263.

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4

CXCR5 Expression by PBMC

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Freshly isolated PBMCs were incubated with recombinant human CXCL-13 (R&D Systems) at 37°C or 4°C and analyzed for CXCR5 surface expression by FACS.

Boswell K.L., Paris R., Boritz E., Ambrozak D., Yamamoto T., Darko S., Wloka K., Wheatley A., Narpala S., McDermott A., Roederer M., Haubrich R., Connors M., Ake J., Douek D.C., Kim J., Petrovas C, & Koup R.A. (2014). Loss of Circulating CD4 T Cells with B Cell Helper Function during Chronic HIV Infection. PLoS Pathogens, 10(1), e1003853.

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5

Chemotaxis Assay for CXCR5+ T and B Cells

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Chemotaxis assays were carried out as described previously45 (link),51 (link), using 96-well transwell plates with 5 μm pores (Corning). CXCR5⁺ CD8⁺, CD4⁺ T cells and CD19⁺ B lymphocytes as well as CXCR5^- CD3⁺ T cells were sorted from patient PBMCs and rested overnight in complete medium. 2.5 x 10⁴ cells were seeded onto upper wells in 100 μl medium. The bottom wells contained 235 μl PBS with or without 1 μg/ml recombinant human CXCL13 (R&D). After 3 hrs, 50 μl Precision Count Beads™ (Biolegend) were added and transmigrated cells were counted by flow cytometry. Cell numbers were calculated using the following formula: absolute cell count = cell count / bead count x total bead concentration. The chemotactic index was calculated as the ratio of transmigrated CXCR5⁺ cells over transmigrated CXCR5^- control T cells.

Thommen D.S., Koelzer V.H., Herzig P., Roller A., Trefny M., Dimeloe S., Kiialainen A., Hanhart J., Schill C., Hess C., Savic Prince S., Wiese M., Lardinois D., Ho P.C., Klein C., Karanikas V., Mertz K.D., Schumacher T.N, & Zippelius A. (2018). A transcriptionally and functionally distinct PD-1+ CD8+ T cell pool with predictive potential in non-small cell lung cancer treated with PD-1 blockade. Nature medicine, 24(7), 994-1004.

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Chemotaxis Assay for CXCR5+ T and B Cells

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Chemotaxis assays were carried out as described previously45 (link),51 (link), using 96-well transwell plates with 5 μm pores (Corning). CXCR5⁺ CD8⁺, CD4⁺ T cells and CD19⁺ B lymphocytes as well as CXCR5^- CD3⁺ T cells were sorted from patient PBMCs and rested overnight in complete medium. 2.5 x 10⁴ cells were seeded onto upper wells in 100 μl medium. The bottom wells contained 235 μl PBS with or without 1 μg/ml recombinant human CXCL13 (R&D). After 3 hrs, 50 μl Precision Count Beads™ (Biolegend) were added and transmigrated cells were counted by flow cytometry. Cell numbers were calculated using the following formula: absolute cell count = cell count / bead count x total bead concentration. The chemotactic index was calculated as the ratio of transmigrated CXCR5⁺ cells over transmigrated CXCR5^- control T cells.

Thommen D.S., Koelzer V.H., Herzig P., Roller A., Trefny M., Dimeloe S., Kiialainen A., Hanhart J., Schill C., Hess C., Savic Prince S., Wiese M., Lardinois D., Ho P.C., Klein C., Karanikas V., Mertz K.D., Schumacher T.N, & Zippelius A. (2018). A transcriptionally and functionally distinct PD-1+ CD8+ T cell pool with predictive potential in non-small cell lung cancer treated with PD-1 blockade. Nature medicine, 24(7), 994-1004.

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Chemotaxis Assay for CXCR5+ Lymphocytes

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Six cell subsets, including CXCR5⁺CD19⁺ B, CXCR5⁺CD4⁺ T, CXCR5⁺CD8⁺ T, CXCR5⁻CD4⁺ T, CXCR5⁻CD8⁺ T and CXCR5⁻CD19⁺ B cells were sorted from NPC-derived PBMCs using a MoFlo Astrios (Beckman) instrument under sterile operation. The chemotaxis assay was done according to the manufacturer’s instructions of the chemotaxis kit (Corning) and published literature.26 27 (link) The cells were rested for 12 hours in RPMI1640 medium with 10% FBS supplemented with 2 mM L-glutamine and 100 ng/mL penicillin/streptomycin. Chemotaxis assays were carried out using 48-well transwell plates with 5 µm pores (Corning), according to the manufacturer’s instructions. Each upper well was seeded with 5×10⁴ cells in 100 µL medium. The bottom wells contained 235 µL PBS with or without 5 µg/mL recombinant human CXCL13 (R&D). After 3 hours, 50 µL Precision Count Beads (BioLegend) were added and transmigrated cells were counted by flow cytometry. Cell numbers were calculated using the following formula: absolute cell count=cell count/bead count×total bead concentration. The chemotactic index was calculated as the ratio of transmigrated CXCR5⁺ cells to transmigrated CXCR5⁻ control cells. The chemotaxis index of CXCR5⁻ subsets was set as ‘1’.

Li J.P., Wu C.Y., Chen M.Y., Liu S.X., Yan S.M., Kang Y.F., Sun C., Grandis J.R., Zeng M.S, & Zhong Q. (2021). PD-1+CXCR5−CD4+ Th-CXCL13 cell subset drives B cells into tertiary lymphoid structures of nasopharyngeal carcinoma. Journal for Immunotherapy of Cancer, 9(7), e002101.

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Recombinant human cxcl13

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7 protocols using recombinant human cxcl13

Transwell Assay for B Cell Migration

Culturing Human Renal Mesangial Cells

Effect of CXCL13 on HRMC Proliferation

CXCR5 Expression by PBMC

Chemotaxis Assay for CXCR5+ T and B Cells

Chemotaxis Assay for CXCR5+ T and B Cells

Chemotaxis Assay for CXCR5+ Lymphocytes

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